ABSTRACT Several recent technical advances in gene engineering technologies are improving both the safety and efficiency with which human hematopoietic stem cells (HSC) can be engineered. In particular, the new class of targeted nucleases, which includes zinc finger nucleases (ZFNs), TALENs and the CRISPR/Cas9 system, are allowing the precise modification of genetic loci and are therefore being embraced for human gene therapy applications. For HIV/AIDS, the first-in-man use of ZFNs is being explored in a strategy based on disruption of the CCR5 co-receptor gene in patients' own T cells. A similar HSC based trial is expected to follow shortly, providing the possibility of more long-term effects from this therapy. However, CCR5 disruption strategies alone may be limited by the fact that (i) cells with only a single CCR5 allele disrupted by the ZFNs will not become resistant to HIV, and (ii) there will be no resistance to dual or CXCR4-tropic viruses. To address these issues and to develop the next generation of therapies for HIV/AIDS based on these remarkable gene editing tools, we are now exploiting their abilities to promote precise gene editing or gene insertion events. This is possible because nuclease-induced DNA breaks can be repaired by homology directed repair (HDR) pathways, where genetic information is copied from a homologous `donor sequence'. Such donors can be designed to result in gene editing events, or to promote the insertion of new genetic material at a highly- specific location. We have recently bypassed a significant bottleneck that had limited the use of this technology in human HSC and can now achieve HDR-mediated gene editing at high levels. We will use this capability to expand the use of targeted nucleases beyond CCR5 gene knockout, by developing new approaches for HIV gene therapy based on either the in situ editing of endogenous human restriction factors, or the precise knock- in of broad spectrum anti-HIV genes at defined loci.